1. Field of the Invention
The present invention is directed to a DAP-kinase related protein.
2. Description of the Related Art
One of the factors which determines the proliferation state of cells is the balance between the growth-promoting effects of proto-oncogenes and the growth-constraining effects of tumor-suppressor genes. One mechanism by which tumor-suppressor genes exert their growth-constraining effect is by inducing a cell to undergo a physiological type of death. Such a controlled cell death is evident in a multitude of physiological conditions including metamorphosis, synaptogenesis of neurons, death of lymphocytes during receptor repertoire selection, and controlled homeostasis in the bone marrow and other proliferative tissues, etc. This cell death is regulated by the interaction of the cell with other cells or with cell products, for example through the activity of suitable cytokines.
Growth-inhibiting cytokines have a double effect on the target cell. They can either inhibit the proliferation of the cell and/or give rise to cell death. To date, blockage or activation of expression of known tumor-suppressor genes was shown to counteract or enhance, respectively, cytokines inhibition of cells growth (Kimchi, 1992) but did not have any effect on the death-promoting action of cytokines. For example, the growth inhibitory response to cytokines, such as TGF-β, was markedly reduced by the inactivation of the Rb gene, or the response to IL-6 was enhanced by introducing activated p53 genes (Pietenpol et al, 1990; Levy et al, 1993).
Apoptosis is a genetically controlled cell death process which is important in various developmental stages, as well as for cell maintenance and tissue homeostasis (Jacobson et al., 1997). During the last few years, many of the key players in this process have been identified, including receptors, adaptor proteins, proteases, and other positive and negative regulators (Green et al., 1998; White, 1996). One of the positive mediators of apoptosis, which has been cloned by the present inventors, is DAP-kinase (Deiss et al., 1995). This protein was discovered by a functional approach to gene cloning, based on transfections of mammalian cells with anti-sense cDNA libraries and subsequent isolation of death-protective cDNA fragments (Deiss et al., 1995; Deiss et al., 1991; Kimchi, 1998; Kissil et al., 1998; Levy-Strumpf et al., 1998). The anti-sense cDNA of DAP-kinase protected HeLa cells from interferon-gamma-induced cell death, and this property served as the basis for its selection.
DAP-kinase is a calcium/calmodulin-regulated 160 kDa serine/threonine protein kinase associated with actin microfilaments (Deiss et al., 1995; Cohen et al., 1997). Its structure contains at least two additional domains that might mediate interactions with other proteins: ankyrin repeats, and a typical death domain located at the C-terminal part of the protein (Deiss et al., 1995; Cohen et al., 1997). Overexpression of DAP-kinase in various cell lines results in cell death, and this death-promoting effect of DAP-kinase depends on at least three features: the catalytic activity, presence of the death domain, and the correct intracellular localization (Cohen et al., 1997; Cohen et al., 1999). Several independent lines of evidence proved that DAP-kinase is involved in apoptosis triggered by different external signals, including interferon-γ, TNF-α, activated Fas receptors, and detachment of cells from the extracellular matrix (Deiss et al., 1995; Cohen et al., 1997; Cohen et al., 1999; Inbal et al., 1997). A tumor suppressive function was recently attributed to the DAP-Kinase, coupling the control of apoptosis to metastasis (Inbal et al., 1997).
So far, only a few serine/threonine kinases were implicated in the regulation of programmed cell death, either as death-promoting and death-protecting proteins (Anderson, 1997; Bokoch, 1998). One such candidate is the JNK/SAPK (Basu et al., 1998). In one example, it was shown to mediate apoptosis induced by detachment from extracellular matrix (named anoikis) (Cardone et al, 1997). In this system, the JNK pathway is activated by MEKK-1, whose kinase activity is stimulated by caspase cleavage (Cardone et al., 1997). JNK may antagonize BCL-2 anti-apoptotic effects by phosphorylation (Park et al., 1997; Maundrell et al., 1997).
Another serine/threonine kinase is RIP, which like DAP-Kinase also possesses the death domain. RIP was shown to positively mediate apoptosis in cell cultures (Stanger et al., 1995). However, in vivo studies in RIP-deficient mice demonstrated its ability to exert anti-apoptotic effects by mediating the TNF-α-induced TNF-β activation (Kelliher et al., 1998). Other RIP members, RIP2 and RIP 3 were also recently identified and shown to possess pro-apoptotic effects (McCarthy et al., 1998; Sun et al., 1998; Yu et al., 1999).
Among the negative regulators of apoptosis is the protein kinase AKT. This protein was shown to phosphorylate BAD and thereby to prevent it from complexing and blocking the anti-apoptotic activity of BCL-XL (Datta et al, 1997; del Peso et al., 1997). AKT was also recently shown to phosphorylate pro-caspase-9, thus blocking its normal processing (Cardone et al., 1998).
Recently, the isolation and characterization of novel kinase members, homologous in their catalytic domains to DAP-kinase, was reported (Kawai et al., 1998; Kogel et al., 1998; Sanjo et al., 1998). One protein, named ZIP-kinase, was found to be 80% identical to DAP-kinase within the kinase domain, yet it lacks the CaM-regulatory domain and the other domains and motifs characteristic of DAP-kinase. Zip-kinase contains a leucine zipper domain at the C-terminus and is localized to the nucleus (Kawai et al., 1998; Kogel et al. 1998). The activation of ZIP kinase occurs by a different mechanism involving homo-dimerization, mediated by its leucine zipper domain. However, unlike DAP-kinase, ZIP-kinase is a nuclear protein, which instead of being regulated by a calmodulin-binding domain, is activated by homo-dimerization of its leucine-zipper motifs (Kogel et al., 1998). Another two less conserved nuclear proteins, DRAK1 and DRAK2, which are closely related to each other, and which share 50% identity with the kinase domain of DAP-kinase, were also recently characterized. Like ZIP-kinase, the DRAK1 and DRAK2 proteins also lack the CaM-regulatory domain. The overexpression of these two proteins in NIH3T3 cells induces some morphological changes associated with apoptosis, dependent on the functionality of their kinase domain (Sanjo et al., 1998). Together these kinases form a novel subfamily of serine/threonine kinases, as is evident from multiple sequence and phylogenetic analysis (Inbal et al., 1999).
Ectopic expression of the three wild type kinases, but not their catalytically inactive mutants, induced morphological changes characteristic of apoptosis (Kawai et al., 1998; Sanjo et al., 1998). Yet, in the case of ZIP-Kinase, these results are still controversial (Kogel et al., 1998).
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